The interaction between catecholamines and insulin in regulating glucose transport in isolated rat adipose cells has been evaluated. In the presence of insulin, both isoproterenol and adenosine deaminase alone inhibit glucose transport activity - 25%. However, only the latter is accompanied by a corresponding decrease in the insulin-stimulated concentration of plasma membrane glucose transporters. Together, isoproterenol and adenosine deaminase inhibit insulin-stimulated glucose transport activity - 75%. However, these agents decrease the insulin-stimulated concentration of plasma membrane glucose transporters only about - 45%. These results suggest that catecholamines counterregulate insulin-stimulated glucose transport in rat adipose cells through a cAMP-mediated mechanism, but only in part by modulating the translocation of glucose transporters. The mechanism of isoproterenol action has been further examined by measuring the protein kinase activity ratio, a marker of intracellular cAMP concentrations. l) Adenosine deaminase alone decreases insulin-stimulated glucose transport activity by 30% without a change in the protein kinase activity ratio. Isoproterenol alone increases the protein kinase activity ratio without a change in insulin-stimulated glucose transport activity. Thus, under these conditions, changes in glucose transport activity do not correlate with the protein kinase activity ratio. However, 2) adenosine deaminase and isoproterenol in combination act synergistically to markedly increase the protein kinase activity ratio and markedly inhibit glucose transport activity. In conclusion, the counterregulation of insulin-stimulated glucose transport activity by isoproterenol appears to comprise both cAMP-independent and -dependent mechanisms.